The rearrangement of immunoglobulin (Ig) genes allows B cells to generate a wide variety of antibodies. The random nature of recombinant process is beneficial because it creates a near infinite number of antibodies, but some of these antibodies recognize self-antigens and therefore have the potential of being pathogenic. Several central (bone marrow) and peripheral mechanisms have been shown to regulate autoantibodies in transgenic mice including deletion, receptor editing, anergy, and ignorance. However, there is little understanding of autoantibody production and regulation in healthy humans, or how this breaks down in autoimmune conditions like systemic lupus erythematosus (SLE). We have developed an efficient system for enumerating autoantibodies cloned from single human B cells in different stages of development. We have found that the majority of all antibodies expressed by early immature B cells of healthy humans are autoreactive, poly-reactive, or anti-nuclear antibodies. Autoantibody-producing cells were removed from the circulation at two discreet checkpoints, the first in the bone marrow and the second in the periphery. We also describe a striking increase of autoantibody producing B cells in the mature naive B cell compartment of three untreated newly diagnosed SLE patients. The object of this proposal is to investigate where B cell tolerance breaks down in systemic autoimmunity. It will examine the efficiency of central B cell tolerance, peripheral B cell tolerance prior to antigen exposure, and the role of peripheral antigen-driven selection in shaping the autoantibody repertoire in systemic autoimmunity. Immunoglobulin heavy and light chain genes from single CD19+CD10+lgM+CD27- (new emigrant), CD19+CD10-lgM+CD27- (mature naive) and CD19+CD10-lgM +CD27+ (IgM memory) B cells from peripheral blood of SLE patients will be cloned, expressed, and tested for reactivity against a panel of self- and non-self antigens. This project will enumerate autoreactive cells in different B cell compartments in SLE, and thereby establish a relationship between repertoire, autoreactivity, and a break in self-tolerance. Patients will be followed up in the course of their treatment, and follow up samples will be examined to assess the effect of therapy on B cell tolerance alterations in SLE.